Tanshinone IIA pretreatment protects free flaps against hypoxic injury by upregulating stem cell-related biomarkers in epithelial skin cells

BACKGROUND: Partial or total flap necrosis after flap transplantation is sometimes encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. The purpose of this study was to determine whether Tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. METHODS: Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for 2 weeks. Cell Counting Kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Polymerase chain reaction and western blot analysis were used to assess the expression of β-catenin, vascular endothelial growth factor (VEGF), sex determining region Y-box 2 (SOX2), OCT4 (also known as POU domain class 5 transcription factor 1), Nanog, and glycogen synthase kinase-3 beta (GSK-3β) in TSA-treated cells. In other experiments, after mice were pretreated with TSA for 2 weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was conducted to examine Wnt signaling as well as stem cell- and angiogenesis-related biomarkers in epithelial tissue in vivo. RESULTS: Epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2, Nanog, and OCT4 were also higher in TSA-pretreated epithelial cells than in control cells. In the reproducible ischemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and VEGF and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. CONCLUSIONS: TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers

In vitro and in vivo assessment of biocompatibility of AZ31 alloy as biliary stents: a preclinical approach

IntroductionBiomaterial technology due to its lack of or minimal side effects in tissues has great potential. Traditionally biomaterials used were cobalt-chromium, stainless steel and nitinol alloys. Biomaterials such as magnesium (Mg) and zinc (Zn) have good biocompatibility and consequently can be a potential material for medical implants. To date, the effects of AZ31 alloy stent on cell apoptosis are still unclear. The current investigation was designed to determine the effect of AZ31 alloy stent on necrosis and apoptosis of common bile duct (CBD) epithelial cells.Material and methodsWe experimented with application of different concentrations of AZ31 alloy stent to primary mouse extrahepatic bile epithelial cells (MEBECs) and estimated the effect on apoptosis and necrotic cells. Apoptosis and pro-apoptosis expression were estimated through real-time PCR. For in vivo protocol, we used rabbits, implanted the AZ31 bile stent, and estimated its effect on the CBD. AZ31 (40%) concentration showed an effect on the apoptotic and necrotic cells.ResultsReal-time PCR revealed that AZ31 (40%) concentration increased the apoptotic genes such as NF-κB, caspase-3, Bax and Bax/Bcl-2 ratio as compared to the control group. In the in vivo experiment, AZ31 alloy stents were implanted into the CBD and showed an effect on the alteration the hema¬tological, hepatic and non-hepatic parameters.ConclusionsTo conclude, it can be stated that AZ31 induces apoptosis via alteration in genes including nuclear factor kappa-B (NF-κB), caspase-3, Bax and Bax/Bcl-2 ratio and improved the hematological, hepatic and non-hepatic parameters.</jats:sec

Tanshinone IIA Pretreatment Renders Free Flaps against Hypoxic Injury through Activating Wnt Signaling and Upregulating Stem Cell-Related Biomarkers

Partial or total flap necrosis after flap transplantation is sometimes clinically encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. In this study, we determine whether tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for two weeks. Cell counting kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Then, Polymerase chain reaction and Western blot analysis were used to determine the expression of β-catenin, GSK-3β, SOX2, and OCT4 in TSA-treated cells. In vivo, after mice were pretreated with TSA for two weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was also conducted to examine the related biomarkers mentioned above. Results show that epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2 and OCT4 controlled by Wnt signaling were also found higher in TSA pretreated epithelial cells. In the reproducible ischaemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. The results show that TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers

Comparative Transcriptomics Unveils Pathogen-Specific mTOR Pathway Modulation in Monochamus alternatus Infected with Entomopathogenic Fungi

Pine wilt disease (PWD), transmitted by Monochamus alternatus (JPS), poses a severe threat to global pine forests. Although the entomopathogenic fungi Beauveria bassiana (Bb) and Metarhizium anisopliae (Ma) represent environmentally friendly biocontrol alternatives, their practical application is limited by inconsistent field performance and an incomplete understanding of host&ndash;pathogen interactions. We employed dual RNA-seq at the critical 48 h infection time point to systematically compare the transcriptional responses between JPS and Bb/Ma during infection. Key findings revealed distinct infection strategies: Bb preferentially induced autophagy pathways and modulated host carbohydrate metabolism to facilitate nutrient acquisition, triggering corresponding tissue degradation responses in JPS. In contrast, Ma primarily co-opted host amino acid and sugar metabolic pathways for biosynthetic processes, eliciting a stronger immune defense activation in JPS. Notably, the mTOR signaling pathway was identified as a key regulator of the differential host responses to various entomopathogenic fungi. Further functional validation-specifically, the application of a chemical inhibitor and RNAi targeting mTOR in JPS-confirmed that mTOR inhibition selectively enhanced Bb-induced mortality in JPS without affecting Ma virulence. Our findings reveal the molecular determinants of host&ndash;pathogen specificity in PWD biological control and indicate that mTOR regulation could serve as an effective strategy to improve fungal pesticide performance